Biology Reference
In-Depth Information
(do not disturb the interface and recentrifuge if necessary).
Mix by inversion.
5. Centrifuge at 13,000 ×
g
during 5 min at room temperature.
Discard the supernatant and air dry the pellet.
6. Resuspend in 50
μ
l of TE.
7. Use 2-5
l of the extracted yeast DNA to transform the appro-
priate
Escherichia coli
strain (for example DH5
μ
) to recover the
plasmid from the cDNA library. If using a bait plasmid that con-
tains resistance to kanamycin (pGBKT7) the prey plasmid for
each positive clone will be easily isolated by plating the bacteria
transformation into ampicillin plates (all GAL4 prey plasmids
contain resistance to ampicillin). Select at least six transformed
colonies. Extract the plasmids and carry out a restriction analy-
sis, which will determine whether all the recovered clones from
each positive yeast colony contain the same cDNA.
α
1. Co-transform yeast cells with the bait vector and the recovered
cDNA library plasmid following the protocol described in
Subheading
3.1
.
2. Transfer the yeast colonies one by one onto plates of CM.
Make small streaks of 2-3 mm in length of each colony in two
plates. Incubate at 30 °C overnight. Store one of the plates at
4 °C and use the other to analyze the interactions.
3. Collect the yeast from one streak using a sterile toothpick and
resuspend them in 100
Confi rm the Interaction
l of deionized sterile water placed in
1.5 ml microcentrifuge tubes.
4. Add 5
μ
l of each suspension on plates with the following media:
(a) IM-HS to select for the induction of ADE2 and HIS3.
(b) IM-MS to select for the induction of ADE2.
(c) IM-LS to select for the induction of HIS3.
(d) CM to check cell growth.
5. Invert the plates and incubate them at 30 °C until colonies
appear (3-5 days).
μ
Growth will confi rm the result obtained in the screening, then
cDNA clones will be them sequenced using the appropriate prim-
ers (OPACT-1: ATGATGAAGATACCCC; OPACT-2: TTGAA
GTGAACTTGCG, can be used for prey clones in pGADT7).
Many of the clones identifi ed in the yeast two-hybrid screenings
may be false positives due to rearrangement of the bait-vector, self-
activation of the reporter genes or a nonspecifi c interaction with
the prey-protein. Co-transformation with the rescued prey-vectors
and the bait protein should be performed to confi rm the interaction.
Control for nonspecifi c interactions must be also included, so plas-
mids expressing the bait empty vector or expressing non-related
Determining the Specifi city
of an Interactor
